Endogenous T-cell Research Articles

Application of Genome-Editing Technologies for Off-the-Shelf T Cell-Based Cancer Immunotherapy

: Genetically, engineered T-cell therapy is a personalized treatment that has demonstrated considerable therapeutic potential for solid tumors and hematopoietic cancers. However, endogenous T-cell receptors (TCRs) on the surface of engineered T cells hamper their application in the allogeneic settings by inducing graft-versus-host disease, where endogenous TCRs on the surface of engineered T cells respond to the recipient’s tissues. Since the cause of this allogeneic response is the TCR complex on the surface of the engineered T cells, preventing the expression of TCR components on the surface of these cells is a promising strategy to address this challenge. This review discusses the production of allogeneic chimeric antigen receptor T cells using genome-editing methods.

Development of a B-cell maturation antigen-specific T-cell antigen coupler receptor for multiple myeloma

Background aimsT cells engineered with synthetic receptors have delivered powerful therapeutic results for patients with relapsed/refractory hematologic malignancies. The authors have recently described the T-cell antigen coupler (TAC) receptor, which co-opts the endogenous T-cell receptor (TCR) and activates engineered T cells in an HLA-independent manner. Here the authors describe the evolution of a next-generation TAC receptor with a focus on developing a TAC-engineered T cell for multiple myeloma. MethodsTo optimize the TAC scaffold, the authors employed a bona fide antigen-binding domain derived from the B-cell maturation antigen-specific monoclonal antibody C11D5.3, which has been used successfully in the clinic. The authors first tested humanized versions of the UCHT1 domain, which is used by the TAC to co-opt the TCR. The authors further discovered that the signal peptide affected surface expression of the TAC receptor. Higher density of the TAC receptor enhanced target binding in vitro, which translated into higher levels of Lck at the immunological synapse and stronger proliferation when only receptor–ligand interactions were present. ResultsThe authors observed that the humanized UCHT1 improved surface expression and in vivo efficacy. Using TAC T cells derived from both healthy donors and multiple myeloma patients, the authors determined that despite the influence of receptor density on early activation events and effector function, receptor density did not impact late effector functions in vitro, nor did the receptor density affect in vivo efficacy. ConclusionsThe modifications to the TAC scaffold described herein represent an important step in the evolution of this technology, which tolerates a range of expression levels without impacting therapeutic efficacy.

CRASH-IT Switch Enables Reversible and Dose-Dependent Control of TCR and CAR T-cell Function.

Adoptive transfer of genetically modified or donor-derived T cells can efficiently eradicate human tumors but is also frequently associated with major toxicity. There are several switches that can be used to kill the infused cell pool in the case of major toxicity, but the irreversible nature of these suicide switches means that the therapeutic effect is lost when they are used. To address this issue, we engineered a small-molecule responsive genetic safety switch that in the absence of drug robustly blocked cytotoxicity and cytokine expression of primary human T cells. Upon administration of drug, T-cell functions were restored in a reversible and titratable manner. We showed that this T-cell switch was universal, as it could be combined with endogenous or transduced T-cell receptors (TCR), as well as chimeric antigen receptors. The modular nature of the Chemically Regulated - SH2-delivered Inhibitory Tail (CRASH-IT) switch concept, in which inhibitory domains are brought to activating immune receptors in a controlled manner, makes it a versatile platform to regulate the activity of cell products that signal through immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors.

Bispecific Anti-CD123 x Anti-CD3 Adaptir™ Molecules APVO436 and APVO437 Have Broad Activity Against Primary Human AML Cells In Vitro

Background: The IL-3 receptor CD123 is an attractive target for antibody-based therapies in acute myeloid leukemia (AML) because of its frequent expression on AML blasts and its reported overexpression on leukemic stem cells (LSCs) as compared to normal hematopoietic tissues. Previously, two CD123 x CD3 bispecific targeting ADAPTIR constructs (APVO436 and APVO437) have been shown to bind CD123 with high affinity, engage T-cells and, in the presence of effector T-cells, cause cytotoxicity in CD123-expressing cell lines as well as in an in vivo xenograft model. Here, we further investigated the determinants of anti-tumor efficacy of these constructs in human AML.Materials and Methods: Frozen aliquots of Ficoll-isolated mononuclear cells from peripheral blood or bone marrow specimens were obtained from adults with AML. CD123 expression on myeloblasts and cells lines and the percentage of endogenous CD3+ T-cells in AML specimens were quantified by flow cytometry. To determine specific cytotoxicity, cell lines or primary AML cells were incubated in culture medium containing various concentrations of ADAPTIR constructs APVO436 and APVO437. Exogenous T-cells isolated from healthy volunteers were labeled with CellVue membrane dye and added at different effector:target (E:T) cell ratios. After 48 hours, cell numbers and drug-induced cytotoxicity, using DAPI to detect non-viable cells, were determined by flow cytometry; target AML cells were identified by forward/side scatter properties and negativity for CellVue dye. Specific cytotoxicity was calculated as: 100 x (%DAPItreated - %DAPIcontrol)/(100-%DAPIcontrol) and are presented as mean±SEM of all samples tested. T-cell activation, proliferation and depletion of CD123+ cell populations in normal and AML specimens were assessed using multi-color flow cytometry.Results: We examined the ability of APVO436 and APVO437 to induce cytotoxicity in primary human AML specimens. Of 20 samples tested, 10 had both >60% viability on thaw and >40% viability after 48 hours in cultures and were included for further analysis. The median percentage of myeloid blasts and CD3+ T-cells in the analyzed specimens was 78.5% (range 52-91.7%) and 2.95% (range 0.3-8.8%). With no exogenous T-cells, APVO436 and APVO437 induced modest cytotoxicity; at 1000 pM, APVO436 and APVO437 induced specific cytotoxicity of 6.8±3.8% and 3.9±3.6%, respectively. There was no statistically significant correlation between the percentage of endogenous T-cells present in the sample and the amount of cytotoxicity in the absence of exogenous T-cells. The addition of healthy donor T-cells increased cytotoxicity in a E:T-dependent manner (p <0.0001 for both APVO436 and APVO437) and increasing drug concentrations also increased cytotoxicity in a statistically-significant fashion (p <0.0001 for both APVO436 and APVO437). There was no consistent linear relationship between MFICD123 of blasts (MFICD123, arbitrary units) and cytotoxicity. Significant cytotoxicity was achieved even at the lowest levels of blast CD123 expression (MFICD123 range among patient samples: 349-3423). Inclusion of all 20 samples in cytotoxicity analysis did not qualitatively change results. APVO436 induced T-cell activation in both normal and AML samples at 24 hours as assessed by quantification of CD25 and CD69 expressing T-cells. T-cell activation was accompanied by proliferation of T-cells in normal and AML specimens at 96 hours in the presence of APVO436. APVO436 induced dose dependent depletion of CD123+ cells which was evident at 24 hours in both normal and AML samples.Conclusion: APVO436 and APVO437 cause target cell cytolysis in a dose-dependent, target-antigen-dependent, and E:T-dependent manner. Broad activity was seen in primary human AML samples and both constructs were effective in inducing cytotoxicity across a range of blast CD123 expression levels, including specimens with very limited CD123 expression. Endogenous T-cell activation and proliferation accompanied by CD123+ cell depletion demonstrate similar activity of anti-CD123 x anti-CD3 ADAPTIRs in both normal and AML subject samples. These data are supportive of further investigation of anti-CD123 x anti-CD3 ADAPTIR molecules as a potential treatment option for AML. DisclosuresGottschalk:Aptevo Therapeutics: Employment, Equity Ownership. Comeau:Aptevo Therapeutics: Employment, Equity Ownership. Hoyos:Aptevo Therapeutics: Employment, Equity Ownership. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding.

Evaluation of T Cell Compartment By Mass Cytometry Reveals Distinct Patterns of Expression of Exhaustion Markers in Chronic Lymphocytic Leukemia

T cell exhaustion is characterized by the coordinated expression of a series of inhibitory receptors such as programmed death-1 (PD-1), 2B4, CD160 and TIGIT, arising in the context of chronic infection and cancer. It is a progressive process characterized by the stepwise acquisition of inhibitory receptors and a corresponding decrease in effector function. The recent interest in strategies to invigorate tumor specific T cells and the use of checkpoint inhibitors have provided a paradigm-shift in cancer treatment. However, not all patients achieve a favorable clinical response to checkpoint blockade. Thus, there is an urgent need to understand the underlying mechanisms that lead to immunotherapy resistance.To this end, we designed a Cytometry by Time-of-Flight (CyTOF) panel of 40 monoclonal antibodies recognizing maturation markers, exhaustion markers, homing and chemokine receptors, and performed a detailed single cell analysis of the T cell repertoire in peripheral blood samples collected from 46 untreated CLL patients and 21 age-matched healthy controls. CD8+ T-cells from patients with CLL had significantly higher expression of a number of checkpoint molecules and differentiation markers, including 2B4 (75.7%±2.2% vs 46.9%±3.6%, p<0.0001), TIGIT (59.3%±2.5% vs 41.05%±2.7%, p<0.0001), PD-1 (42.8%±2.6% vs 32.4%±2.07% p<0.05), CD160 (33.6%±2.2% vs 25.4%±2.7% p<0.05), CD57 (49.1%±3.2% vs 26.4%±3.2%, p<0.0001) and KLRG1 (74.7%±2.9% vs 46.1%±5.2%, p:0.0005) compared to healthy controls. Similarly, CD4+ T cells in CLL expressed significantly higher levels of inhibitory molecules, including TIGIT (28.6%±2.07% vs 18.1%±0.7% p=0.0005), CD39, a marker of exhaustion involved in the generation of the immunosuppressive molecule adenosine (13.7%±2.4% vs 5.5%±0.6% p<0.005) and PD-1 (43.5%±2.3% vs 32.3%±1.4%, p<0.05) compared to healthy donors.To further interrogate the CD8+ and CD4+ T-cell compartments in CLL, we performed high-dimensional visualization and unsupervised hierarchical clustering of the CyTOF data. The majority of CD8+ T cells from CLL patients expressed multiple checkpoint molecules per cell when compared to healthy donors, with co-expression of 2 receptors seen in 23.6%±1.1% vs. 18.6%±1.2%; p<0.05, 3 receptors in 31.07%±1.6% vs. 17.73%±1.4%; p<0.0001 and 4 inhibitory receptors in 13.5%±1.4% vs. 6.7%±0.9%; p:0.0005, respectively. A significantly greater proportion of CD4+ and CD8+ T-cells co-expressed TIGIT and PD-1. Interestingly, the inhibitory molecule CD160 was always co-expressed in conjunction with 2B4, TIGIT, CD57 and/or KLRG1, with very few single CD160 expressing CD8+ T-cells and negligible expression in the absence of other checkpoint molecules, indicating that CD160 expression is a late event during T cell exhaustion in CLL.Focusing on key T cell subsets expressing combinations of inhibitory receptors allowed greater characterization of CLL T cells and led us to conclude that CD4+ T cells expressing inhibitory receptors are exhausted while CD8+ T cells are both exhausted and senescent, based on high expression of CD57 and KLRG1. The exhausted/senescent CD8+ T cell compartment in CLL was heterogeneous and was characterized by altered and reduced expression of chemokine and homing receptors, including CCR6, CCR4, CCR2 and CXCR3. Notably, CD8+ exhausted/senescent T cells expressed significantly reduced levels of the co-stimulatory molecules, CD28 and CD27, with reduced expression of homing receptors and activating receptors. In contrast, CD127 expression was significantly higher on exhausted/senescent T cells, suggesting that IL-7 could be important in the maintenance of this subset.Taken together, our findings indicate a remarkable heterogeneity in the expression patterns of inhibitory molecules on CD8+ and CD4+ T-cells in CLL. On a per cell basis, CLL CD8+ T cells expressed more inhibitory receptors compared to healthy controls, suggesting that certain patients may benefit from combinational use of checkpoint molecules. In-depth dissection of the CD8 T cell compartment revealed a dominantly senescent/exhausted T cell profile with limited cell proliferation capacity, suggesting that checkpoint inhibition is less likely to invigorate the endogenous antitumor T-cell response in such patients. DisclosuresWierda:The University of Texas MD Anderson Cancer Center: Employment; Sanofi: Consultancy, Honoraria; Juno: Research Funding; Kite: Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; Emergent: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Acerta: Research Funding; Karyopharm: Research Funding. Jain:Genentech: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Verastem: Research Funding; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding. Thompson:Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees.

A phase 1 dose escalation and cohort expansion study of the safety and efficacy of allogeneic CRISPR-Cas9–engineered T cells (CTX110) in patients (Pts) with relapsed or refractory (R/R) B-cell malignancies (CARBON).

TPS7570 Background: Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL), and although &gt; 50% of pts achieve long-term remission with first-line therapy, pts with R/R disease as well as those with R/R grade 3b follicular lymphoma (FL), double-, or triple-hit high-grade lymphomas have poor long-term outcomes (Crump 2017; Kahl 2016; Jain 2012). Autologous (auto) chimeric antigen receptor (CAR) T cell therapy has provided additional options for pts with R/R disease, but only when leukapheresis and manufacturing prove feasible (Jacobson 2020). Allogeneic (allo) CAR-T cells were designed specifically to address these unmet needs by using healthy donor T cells to produce a readily available product and remove the need for bridging chemotherapy. We are currently investigating the safety and efficacy of CTX110, an allo anti-CD19 CAR-T cell product modified by using CRISPR/Cas9-editing to disrupt the endogenous T-cell receptor (TCR) alpha constant (TRAC) locus in order to remove TCR expression and disrupt β2-microglobulin, which eliminates major histocompatibility complex (MHC) class I expression. Disruption of the TCR should significantly reduce or eliminate risks of graft-versus-host disease and elimination of MHC class I expression may increase CAR-T cell persistence by mitigating CTX110 rejection. In addition, the anti-CD19 CAR transgene construct is precisely inserted into the TRAC locus. Methods: The Phase 1 CARBON trial (NCT04035434) is an open-label, multicenter, global study evaluating the safety and efficacy of CTX110 in pts ≥18 y with R/R DLBCL NOS, double- or triple-hit DLBCL, or transformed or grade 3b FL with ≥2 prior lines of therapy or who are ineligible for/refused prior auto hematopoietic stem cell transplant (HSCT). Pts who received prior auto CAR-T or allo HSCT are excluded. Pts will receive lymphodepleting chemotherapy with fludarabine 30mg/m2 and cyclophosphamide 500mg/m2 for 3 days, followed by CTX110 infusion. In part A, dose escalation will be performed using a 3+3 design. Upon completion of dose finding, the cohort will be expanded to further assess safety signals and efficacy including the primary efficacy endpoint of overall response rate. Key secondary efficacy endpoints include duration of response, progression-free survival, and overall survival. The trial is currently open and enrolling. Clinical trial information: NCT04035434.

CDK4/6 Inhibition Promotes Antitumor Immunity through the Induction of T-cell Memory.

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor-positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. SIGNIFICANCE: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer.This article is highlighted in the In This Issue feature, p. 2355.

Obese microenvironments attenuate the efficacy of endogenous and CD19-directed CAR T-cells

Abstract Background: Hematological malignancies represent the largest class of cancers in pediatric patients. Despite successes in treating primary diseases, patients with relapsed B-cell acute lymphoblastic leukemia (B-ALL) and diffuse large B-cell lymphoma (DLBCL) have significantly lower survival outcomes. This has led to the use of immunotherapies, notably chimeric antigen receptor (CAR) T-cells, to treat relapse. Unfortunately, only 50% respond to CAR-T therapy, with relapse common in less than a year. Objective: Given that obesity is associated with poorer survival outcomes in pediatric patients with B-cell malignancies and promotes a loss of immune homeostasis, we hypothesize that obesity compromises the function of endogenous and engineered T-cells in B-cell malignancies. Results: Using in vitro models of adiposity and diet-induced obesity mouse models to study the impact of obesity on primary T-cell function, we observe defects in proliferation, cytokine production, and the upregulation of cytolytic mediators when cultured in adipocyte but not in bone marrow cell or unconditioned media. The introduction of CD19-direct CARs did not overcome adipocyte-induced defects in T-cell function, which resulted in increased PD-1 surface expression, compromised cytokine production, and defective killing of human CD19-expressing B-ALL cells. Interestingly, human B-ALL cells cultured in adipocyte-secreted factors downregulated CD19, suggesting that adipocytes promote B-ALL pathogenesis by compromising T-cell function and reducing the surface expression of antigens on target cells. Conclusion: Our studies are the first to demonstrate the inhibitory impact of the adipocyte secretome on CAR T-cell function in models of B-ALL.

IFNγ Is Critical for CAR T Cell–Mediated Myeloid Activation and Induction of Endogenous Immunity

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE: Our findings highlight the critical role of IFNγ signaling for a productive CAR T-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.

Interleukin-37 improves T-cell-mediated immunity and chimeric antigen receptor T-cell therapy in aged backgrounds.

Aging‐associated declines in innate and adaptive immune responses are well documented and pose a risk for the growing aging population, which is predicted to comprise greater than 40 percent of the world's population by 2050. Efforts have been made to improve immunity in aged populations; however, safe and effective protocols to accomplish this goal have not been universally established. Aging‐associated chronic inflammation is postulated to compromise immunity in aged mice and humans. Interleukin‐37 (IL‐37) is a potent anti‐inflammatory cytokine, and we present data demonstrating that IL‐37 gene expression levels in human monocytes significantly decline with age. Furthermore, we demonstrate that transgenic expression of interleukin‐37 (IL‐37) in aged mice reduces or prevents aging‐associated chronic inflammation, splenomegaly, and accumulation of myeloid cells (macrophages and dendritic cells) in the bone marrow and spleen. Additionally, we show that IL‐37 expression decreases the surface expression of programmed cell death protein 1 (PD‐1) and augments cytokine production from aged T‐cells. Improved T‐cell function coincided with a youthful restoration of Pdcd1, Lat, and Stat4 gene expression levels in CD4+ T‐cells and Lat in CD8+ T‐cells when aged mice were treated with recombinant IL‐37 (rIL‐37) but not control immunoglobin (Control Ig). Importantly, IL‐37‐mediated rejuvenation of aged endogenous T‐cells was also observed in aged chimeric antigen receptor (CAR) T‐cells, where improved function significantly extended the survival of mice transplanted with leukemia cells. Collectively, these data demonstrate the potency of IL‐37 in boosting the function of aged T‐cells and highlight its therapeutic potential to overcome aging‐associated immunosenescence.

Single-Cell Transcriptomic Analysis Reveals BCMA CAR-T Cell Dynamics in a Patient with Refractory Primary Plasma Cell Leukemia

Chimeric antigen receptor Tcell (CAR-T) therapy has revolutionized the clinical treatment of hematological malignancies due to the prominent anti-tumor effects. B cell maturation antigen (BCMA) CAR-T cells have demonstrated promising effects in patients with relapsed/refractory multiple myeloma. However, the dynamics of CAR-T cell proliferation and cytotoxicity in clinical patients remains unexplored. Here, we longitudinally profiled the transcriptomes of 55,488 Tcells including CAR-T products, CAR-T cells, and endogenous Tcells at the peak and remission phases in a plasma cell leukemia (PCL) patient treated with BCMA CAR-T cells by single-cell transcriptomic analysis. Our results showed distinct CAR-T and endogenous Tcell subsets indicating stage-specific expression in proliferation, cytotoxicity, and intercellular signaling pathways. Furthermore, we found that CAR-T cells at peak phase gradually convert to a highly cytotoxic state from a highly proliferative state along a development trajectory. Moreover, re-analysis of a single cell study from CD8+ CD19 CAR-T confirmed our findings. These commonalities suggest conserved mechanisms for CAR-T treatment across hematological malignancies. Taken together, our current study provides insight into CAR-T cell dynamics during CAR-T therapy and proves that both BCMA CAR-T and CD19 CAR-T have similar transcriptional characteristics, especially at the CAR-T peak phase.

Obesity Attenuates T-Cell Function Which Impacts the Efficacy of T-Cell Based Immunotherapies in Acute Lymphoblastic Leukemia

The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. The global health impact of this trend is concerning given that obesity is a risk factor for developing cancers of varying etiologies. Alarmingly, the survival outcomes for obese patients with cancer are lower than those observed in lean patients. Obesity is characterized by the accumulation of adipocytes which alters drug dynamics and impacts the function of cancer and immune cells in the tumor microenvironment. Obesity-induced immune defects are troubling given the increasing use of immunotherapy in the treatment of malignancies. Here we show that adipocyte-secreted factors upregulate immunosuppressive mechanisms on human B-cell acute lymphoblastic leukemia (B-ALL) cells, attenuate the function of endogenous T-cells, and compromise the efficacy of T-cell based immunotherapies. To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL. We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity. Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells. Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19. Disclosures No relevant conflicts of interest to declare.

Detecting Tumor Antigen-Specific T Cells via Interaction-Dependent Fucosyl-Biotinylation

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive Tcells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive Tcells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific Tcells for studying their biology. Through this interaction-dependent labeling approach, intratumoral TSA-reactive CD4+, CD8+ Tcells, and TSA-suppressive CD4+ Tcells can be detected and separated from bystander Tcells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs, TSA-reactive TILs possess a distinct Tcell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype, TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle, FucoID should have the potential of accelerating the pace of personalized cancer treatment.

Off-the-shelf EBV-specific T-cell Immunotherapy for EBV-associated PTLD.

Off-the-shelf EBV-specific T-cell Immunotherapy for EBV-associated PTLD.

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR

AbstractAnti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Mechanisms of T-Cell Exhaustion in Pancreatic Cancer.

T-cell exhaustion is a phenomenon that represents the dysfunctional state of T cells in chronic infections and cancer and is closely associated with poor prognosis in many cancers. The endogenous T-cell immunity and genetically edited cell therapies (CAR-T) failed to prevent tumor immune evasion. The effector T-cell activity is perturbed by an imbalance between inhibitory and stimulatory signals causing a reprogramming in metabolism and the high levels of multiple inhibitory receptors like programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and Lymphocyte-activation gene 3 (Lag-3). Despite the efforts to neutralize inhibitory receptors by a single agent or combinatorial immune checkpoint inhibitors to boost effector function, PDAC remains unresponsive to these therapies, suggesting that multiple molecular mechanisms play a role in stimulating the exhaustion state of tumor-infiltrating T cells. Recent studies utilizing transcriptomics, mass cytometry, and epigenomics revealed a critical role of Thymocyte selection-associated high mobility group box protein (TOX) genes and TOX-associated pathways, driving T-cell exhaustion in chronic infection and cancer. Here, we will review recently defined molecular, genetic, and cellular factors that drive T-cell exhaustion in PDAC. We will also discuss the effects of available immune checkpoint inhibitors and the latest clinical trials targeting various molecular factors mediating T-cell exhaustion in PDAC.

A Distinct Transcriptional Program in Human CAR T Cells Bearing the 4-1BB Signaling Domain Revealed by scRNA-Seq

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have produced impressive outcomes for the treatment of B cell malignancies, but different products vary in kinetics, persistence, and toxicity profiles based on the co-stimulatory domains included in the CAR. In this study, we performed transcriptional profiling of bulk CAR Tcell populations and single cells to characterize the transcriptional states of human Tcells transduced with CD3ζ, 4-1BB-CD3ζ (BBζ), or CD28-CD3ζ (28ζ) co-stimulatory domains at rest and after activation by triggering their CAR or their endogenous Tcell receptor (TCR). We identified a transcriptional signature common across CARs with the CD3ζ signaling domain, as well as a distinct program associated with the 4-1BB co-stimulatory domain at rest and after activation. CAR Tcells bearing BBζ had increased expression of human leukocyte antigen (HLA) class II genes, ENPP2, and interleukin (IL)-21 axis genes, and decreased PD1 compared to 28ζ CAR Tcells. Similar to previous studies, we also found BBζ CAR CD8 Tcells to be enriched in a central memory cell phenotype and fatty acid metabolism genes. Our data uncovered transcriptional signatures related to costimulatory domains and demonstrated that signaling domains included in CARs uniquely shape the transcriptional programs of Tcells.

A phase Ib study of endogenous SLC45A2-specific cytotoxic T cells for the treatment of patients with metastatic uveal melanoma.

TPS10086 Background: Overall survival (OS) for patients (pts) with advanced uveal melanoma (UM) is poor with a median survival of approximately 12 months. Roughly half of all pts with UM will develop distant metastatic disease despite effective treatment of the primary site. Metastatic UM has a 90% prevalence of liver involvement. Currently, there are no specific FDA-approved treatments for metastatic UM and consensus guidelines recommend participation in a clinical trial. Modern treatments such as checkpoint inhibitors and targeted therapy have less impressive outcomes in UM. Our group has identified peptide epitopes of SLC45A2, a melanosomal transport protein, that is highly expressed in UM and present at very low levels in normal melanocytes. We demonstrated that cytotoxic T cells against SLC45A2 were able to kill HLA-matched UM cell lines. Through the use of enabling technologies, SLC45A2-specific cytotoxic T cells can be isolated and expanded ex-vivo from peripheral blood for use in endogenous T cell (ETC) therapy (a form of adoptive cellular therapy). These activated ETCs can then be infused to traffic to tumor sites. CTLA4 is a T-cell surface protein that binds to B7 with a higher affinity than the costimulatory receptor CD28, providing an inhibitory signal to T-cells. Anti-CTLA4 blockade can divert this inhibition and release the brake on antigen-specific T-cell activation of ETC. We hypothesize that antigen-specific ETCs infused via hepatic artery will be safe and tolerable for UM pts with liver metastasis. Methods: We are conducting a first-in-human clinical trial (NCT03068624 ) of ETC therapy targeting SLC45A2 in combination with anti-CTLA4 in pts with metastatic UM. Pts who express HLA-A*02:01 or A*24:02 undergo apheresis to collect T cells. Their cells then undergo ex vivo cloning and interleukin-21 primed expansion. Hepatic arterial infusion of ETCs will ensure direct localization to the target organ of interest. Conditioning with low-dose cyclophosphamide (300 mg/m2) occurs on Day -2. Hepatic arterial infusion of ETCs on Day 0 is followed by low dose subcutaneous interleukin-2 (IL-2) twice daily for 14 days. The initial dose escalation phase is a 3+3 design with a starting dose level of 3.3 x 109 cells/m2 of ETCs alone. Once the maximum tolerated dose of ETCs is established, the dose expansion phase will include ETCs in combination with anti-CTLA4 (Ipilimumab). The primary objective is to evaluate the safety of this first in human T cell regimen. Secondary objectives are to evaluate the in vivo persistence and anti-tumor efficacy. Clinical trial information: NCT03068624.

Allogeneic CAR T-cells resistant to both T- and NK-cell cytotoxicity

Background & Aim The last years have seen the adoptive transfer of engineered autologous T-cells making great strides in the development of new treatments against cancer. The use of third-party donor-derived T-cells represents an attractive alternative to generate CAR T-cells readily accessible to patients. These off-the-shelf cells derived from a possibly non-HLA-matched donor nevertheless carry the risk of graft-versus-host-disease (GvHD) through the expression of their endogenous T-cell receptors (TCRs). The recent advances in precise genome editing using designer nucleases allowed to mitigate this issue as demonstrated by the molecular remissions observed in patients after infusion of universal TALEN® multiplex gene-edited CAR T-cells. In particular, these universal CAR T-cells were engineered to reduce the risk of graft versus host disease (GvHD) by TALEN® inactivation of TCRαβ. The clinical outcome of CAR T-cell therapies is intimately linked to the ability of effector cells to engraft and proliferate in order to eradicate tumor cells within patients. Although the transient activity of off-the-shelf CAR T-cells represents an important safety feature, the possibility to extend their therapeutic window may be desirable in particular disease indications and after lymphodepleting regimens where the patient's immune system has been restored. Thus, we are developing novel approaches to render these cells less visible to both host T- and NK-cells. Methods, Results & Conclusion The single genetic disruption of the beta-2 microglobulin (B2M) gene, a required component of all MHC class I molecules, promotes resistance to host CD8+ T-cell attack but may trigger NK-cell activation, leading to the rejection of the edited T-cells lacking MHC-I. We therefore further engineered these B2M negative cells via a TALEN®-mediated and B2M gene-specific targeted integration matrix to express inhibitors of NK cytotoxicity. We have identified an NK-cell inhibitor that successfully blocks the so-called NK-cell “missing self-response” in vitro and in vivo. Altogether, the precise TALEN®-mediated inactivation of B2M coupled with insertion of an NK-cell inhibitor enabled the generation of allogeneic CAR T-cells, resistant to host T- and NK-cells, with improved persistence and long-term anti-tumor activity.

Abstract IA07: Hitting the target: T-cell receptors, cross-reactivity, and tumors

Abstract Cancer arises due to an accumulation of genetic alterations, which can lead to the production of mutant proteins that are not expressed by normal cells. Some of these mutant proteins can be processed into short peptides and presented on the cell surface by major histocompatibility complex molecules as neoepitopes and CD8+ T cells can effectively recognize and mount responses against these neoepitopes across various cancer types. However, tumor-specific lymphocytes are generally characterized by an exhausted phenotype, which can in some circumstances be reversed with checkpoint blockade therapies that restore antitumor activity, with those cancer types and individual tumors that bear relatively high mutation burdens showing more robust responses. Using predictive algorithms to identify putative endogenous antitumor T-cell responses across solid tumor types has resulted in an average 2% of predicted neoepitopes associated with a detectable response. This has led to the assumption that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphocytic leukemia (ALL) possess broad pools of tumor-associated neoepitope-specific CD8+ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. We characterized phenotypic and transcriptional profiles of these epitope-specific antitumor CD8+ T cells at the single-cell level and found a heterogeneous population that included many highly functional effector cells. Moreover, we observed immunodominance hierarchies among the neoepitope-specific CD8+ TILs, whereby a majority of tetramer-bound CD8+ T cells were restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in these pediatric ALL tumors despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. These data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens. Citation Format: Paul G. Thomas. Hitting the target: T-cell receptors, cross-reactivity, and tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA07.